Nitric Oxide Production Measurement

The NO production measurements were performed as described previously [8 (link),33 (link),35 (link)]. Briefly, BMMs (1 × 10⁶ cells/well, n = 4 per group) were seeded into 48-well culture plates (Falcon) to prepare the seven experimental groups (control (only BMMs), 0.1 μg/mL of LPS, 1 μg/mL of MP, and 0.5, 1, 10, and 100 μg/mL of TEGO). After 24 h, NO products accumulated in the supernatant were detected using a Griess reagent system (Promega, Madison, WI, USA). Briefly, the supernatant and sulfanilamide were mixed in equal amounts. Each group was measured at a wavelength of 548 nm using a microplate absorbance reader (Bio-Rad, Hercules, CA, USA). The NO concentration in the supernatant fluid was measured with a standard curve generated with sodium nitrite.